JP7467358B2 - In vivo expansion of NK and DC cells that mediate immune responses - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/632,733, filed February 20, 2018, the contents of which are incorporated herein by reference in their entirety.

A method is provided for expanding dendritic (DC) cells and/or natural killer (NK) cells in vivo in a patient by transplanting grafts of stem and progenitor cells cultured with UM171 or an analog derived therefrom.

Immunotherapy is considered a major breakthrough in the field of anticancer therapy, since this approach has demonstrated its effectiveness against chemotherapy-refractory cancers. Although most attempts have focused on antigen-targeted approaches, approaches that utilize innate immunity to fight cancer cells have also been proposed, and natural killer (NK) cells are increasingly being used in the design of anticancer immunotherapies.

NK cells recognize and kill infected or transformed cells without prior sensitization. The cytotoxic activity of NK cells against cancer cells is highly regulated by the balance between activating and inhibitory signals and the education that NK cells receive to distinguish self and non-transformed cells from cancer and infected cells. However, cancer cells can become resistant to NK cell-mediated lysis by downregulating ligands for NK cell activating receptors. To overcome this resistance, it is necessary to stimulate NK cells to enhance their cytotoxic function. Interleukin (IL)-2 and IL-15 are the cytokines most frequently used to enhance the lytic function of NK cells, but their clinical use is associated with high toxicity and side effects that may hinder the effectiveness of NK cell-mediated cytotoxicity against cancer. Natural killer cells play a crucial role in the defense against cancer relapse and infections.

NK cell function can be stimulated by a small number of activated dendritic cells. Dendritic cells (DCs) are the most potent antigen-presenting cells that stimulate both innate and adaptive immune responses, thereby resulting in infection resistance, protective antitumor immunity, and self-tolerance. Their unique intrinsic ability to generate large numbers of high-avidity effector cells, such as cytotoxic T lymphocytes and natural killer cells, makes DCs excellent candidates for cell-based therapy against a variety of hematological malignancies, including cancer, infectious diseases, allergies, and autoimmune diseases. DCs can shape their function based on the self-immune status, which is critical for the balance between immunity and tolerance to maintain homeostasis. In the immune response associated with stem cell transplantation, DCs are involved in the induction of immune tolerance and antitumor immunity.

Several DC subsets are characterized by expressing different repertoires of Toll-like receptors (TLRs) and surface molecules and different sets of cytokines/chemokines, all of which mediate characteristic and specific humoral and/or cellular immune responses. The two main subsets are myeloid DCs (mDCs) and plasmacytoid DCs (pDCs).

mDCs and pDCs respond differently to pathogenic stimuli, with each subset having specialized functions in directing the immune response. mDCs respond to microbial stimuli via TLRs by producing TNF-α and IL-12, whereas pDCs produce large amounts of type I interferon (IFN) in response to bacterial or viral infections, making them key effectors in innate immunity. Recent observations suggest that both pDCs and mDCs are important for inducing antitumor responses and may act synergistically to induce better immunological outcomes.

Allogeneic hematopoietic stem cell (HSC) transplantation is the best available cure for patients with hematological cancers. The risks of graft rejection, tumor recurrence and tumorigenicity remain after stem cell transplantation. Unfortunately, 40% of patients lack a human leukocyte antigen (HLA)-matched donor (related or unrelated). Umbilical cord blood (CB) has unique properties that make it the most attractive alternative donor source of stem cells, including tolerance to HLA mismatches, low incidence of chronic graft-versus-host disease (GVHD) and rapid availability. However, these advantages are countered by limited cell doses (i.e., small cord bank volumes), resulting in delayed or non-engraftment, increased infections, prolonged hospitalization and early mortality.

The allogeneic transplant procedure consists of conditioning treatment (chemotherapy +/- radiation) followed by the infusion of stem cells (the graft) to eradicate any remaining cancer cells.

While bone marrow and stimulated peripheral blood stem cells are collected from HLA-matched related or volunteer unrelated donors, umbilical cord blood (umbilical CB) is donated at birth. The duration of neutropenia directly correlates with the risk of severe infection and transplant-related mortality, being shortest for peripheral blood (14 days), followed by bone marrow (19 days), and longest for CB (26 days).

Allogeneic HSC transplants have a transplant-related mortality rate of up to 40%. The most common complications include acute (approximately 50%) and chronic (approximately 60%) GVHD, which is an immune response by the donor against the recipient, frequently damaging mucous membranes (mouth, eyes), skin, liver, lungs, and intestinal tract. In addition, infections are very common, and graft failure (failure to engraft, approximately 10%) occurs due to a lack of stem cells in the graft and/or destruction of the graft by the recipient's immune system. These complications vary according to four main factors: i) the intensity of the conditioning regimen, ii) the type of graft transfused (bone marrow, peripheral blood, or CB), iii) the degree of HLA incompatibility between the donor and recipient, and iv) the patient's comorbidities. GVHD is often treated with high doses of immunosuppressive drugs, such as corticosteroids, and requires treatment for an average of four to five years. The need for such long-term immunosuppressive therapy further increases the risk of infections, secondary cancers, and drug-related toxicity, all of which dramatically affect the patient's quality of life.

Transplant recipients are in a long-term immunocompromised state. Early T cell recovery depends on peripheral expansion of donor memory T cells. This is followed by maturation of donor stem cell-derived lymphoid precursors into naive T cells in the thymus, a step essential for reconstitution of the polyclonal T cell repertoire. Until robust thymic egress is achieved, CB graft recipients have only naive T cells (in the absence of memory cells) and are therefore unable to fight pathogens, increasing the risk of viral infection during the first few months. Because functional CD4 ⁺ T cells are essential for the generation of mature memory B cells, mature memory B cells are not usually fully reconstituted until 1-2 years after HSC transplantation, and humoral immunity is predominantly recipient-derived during the first year.

Currently, only 6% of available CB units have sufficient cell dose for adults. Therefore, double unit CB transplants in adults are becoming the norm. The ease of engraftment is improved by using two units of umbilical cord because the minimum required TNC (total nucleated cell) dose is lowered to 1.5×10 ⁷ /kg per unit of umbilical cord. Neutrophil engraftment is not improved, but the risk of graft failure is reduced. Early after transplantation, both units of umbilical cord are detectable, but after 3 months only one unit remains. As each CB mounts an immune response to eliminate the other, the increased dose of T lymphocytes and CD34 ⁺ cells determines which cord survives. This immune response is responsible for the high incidence of severe acute GVHD and HSC destruction, explaining why engraftment is delayed despite higher cell doses infused. Moreover, the high cost makes double unit CB unaffordable.

It would therefore be highly desirable to provide a method for stimulating NK cells and/or dendritic cells in vivo after transplantation of a graft has been performed.

1. A method of expanding dendritic (DC) cells, natural killer (NK) cells, or a combination thereof in vivo in a patient, comprising:
a) treating a starting stem cell and/or progenitor cell population with at least one compound of formula I:

or a salt or prodrug thereof,
each Y is independently selected from N and CH;
Z is,
- C.N.,
-C(O)OR1,
-C(O)N(R1)R3,
-C(O)R1, or -heteroaryl (optionally substituted with one or more RA or R4 substituents).
and
wherein when (R1) and R3 are bonded to a nitrogen atom, (R1) and R3 together with the nitrogen atom may form a 3- to 7-membered ring which may contain one or more other heteroatoms selected from N, O and S, and which ring is optionally substituted with one or more RA or R4;
W is
- C.N.,
-N(R1)R3,
-C(O)OR1,
-C(O)N(R1)R3,
-NR1C(O)R1,
-NR1C(O)OR1,
-OC(O)N(R1)R3,
-OC(O)R1,
-C(O)R1,
-NR1C(O)N(R1)R3,
-NR1S(O) _2R1 ,
-benzyl (optionally substituted by 1, 2 or 3 R or R substituents);
-X-L-(X-L)n-N(R1)R3,
-XL-(XL)n-heteroaryl, optionally substituted with one or more RA or R4 substituents attached to either or both of the L and heteroaryl groups;
-XL-(XL)n-heterocyclyl (optionally substituted with one or more RA or R4 substituents attached to either or both of the L and heterocyclyl groups);
-XL-(XL)n-aryl (optionally substituted by one or more R or R substituents);
-X-L-(X-L) _n -NR1RA, or -(N(R1)-L) _n -N ⁺ R1R3R5R6 ^-
and
where n is an integer equal to either 0, 1, 2, 3, 4, or 5;
wherein when R1 and R3 are bonded to a nitrogen atom, R1 and R3 together with the nitrogen atom may form a 3- to 7-membered ring which may contain one or more other heteroatoms selected from N, O and S, and which ring is optionally substituted with one or more RA or R4;
Each X is independently selected from C, O, S, and NR1;
Each L is independently
_-C1-6 alkylene,
_-C2-6 alkenylene,
_-C alkynylene,
-C _3-7 cycloalkylene (which may contain one or more other heteroatoms selected from N, O and S), or -C _3-7 cycloalkenylene (which may contain one or more other heteroatoms selected from N, O and S).
and
wherein the alkylene, alkenylene, alkynylene, cycloalkylene, and cycloalkenylene groups are each optionally independently substituted with one or two R or RA substituents;
Each R1 is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-heterocyclyl,
-aryl,
-heteroaryl, or -benzyl,
wherein the alkyl, alkenyl, alkynyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally substituted with 1, 2 or 3 R or R substituents;
R2 is,
-H,
-C _1-6 alkyl (optionally substituted by one or more RA substituents),
-C(O)R4,
-L-heteroaryl (optionally substituted by one or more R or R substituents);
-L-heterocyclyl (optionally substituted with one or more RA or R4 substituents), or -L-aryl (optionally substituted with one or more RA or R4 substituents).
and
Each R3 is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-heterocyclyl,
-aryl,
-heteroaryl, or -benzyl,
wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally substituted with 1, 2 or 3 R or R substituents;
Each R4 is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-heterocyclyl,
-aryl,
-heteroaryl, or -benzyl,
wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally substituted with 1, 2 or 3 R or R substituents;
Each R5 is independently
-C _1-6 alkyl,
_{-Ci_6} alkylene _{-C2_6} alkenyl (which may contain one or more other heteroatoms selected from N, O and S);
_{-Ci_6} alkylene _{-C2_6} alkynyl (which may contain one or more other heteroatoms selected from N, O and S);
-L-aryl (optionally containing one or more R or R substituents);
-L-heteroaryl (optionally containing one or more R or R substituents);
—C _1-6 alkylene-C(O)O—,
_-C1-6 alkylene-C(O)OR1,
—C _1-6 alkylene-CN,
_-C1-6 alkylene-C(O)NR1R3, where R1 and R3 together with the nitrogen atom may form a 3-7 membered ring which may contain one or more other heteroatoms selected from N, O and S, or _-C1-6 alkylene-OH
and
R6 is,
-halogen,
-OC(O) _CF3 , or -OC(O)R1
and
Each RA is independently
-halogen,
_-CF3 ,
-OR1,
-L-OR1,
_-OCF3 ,
-SR1,
- C.N.,
_-NO2 ,
-NR1R3,
-L-NR1R1,
-C(O)OR1,
-S(O) _2R4 ,
-C(O)N(R1)R3,
-NR1C(O)R1,
-NR1C(O)OR1,
-OC(O)N(R1)R3,
-OC(O)R1,
-C(O)R4,
-NHC(O)N(R1)R3,
-NR1C(O)N(R1)R3, or _-N3
and
Each Rd is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-benzyl, or -heterocyclyl.
optionally with at least one cell expanding factor;
b) expanding the cultured stem and/or progenitor cell population to generate a graft;
and c) transplanting said graft into said patient, thereby expanding DC cells, NK cells, or a combination thereof in said patient.

Also provided is the use of a graft of expanded stem and/or progenitor cells cultured with at least one compound as defined herein, optionally together with at least one cell expansion factor, to expand dendritic (DC) cells, natural killer (NK) cells, or a combination thereof in vivo in a patient.

In one embodiment, expansion of DC cells, NK cells, or a combination thereof stimulates an immune response in the patient.

In another embodiment, expansion of DC cells, NK cells, or a combination thereof in a patient further reduces severe graft-versus-host disease (GVHD), relapse, and/or severe viral infection in said patient.

In one embodiment, the stem and/or progenitor cells are human hematopoietic stem cells (HSCs).

In another embodiment, the hematopoietic stem cells are derived from umbilical cord blood cells, mobilized peripheral blood cells, or bone marrow cells.

In a further embodiment, the hematopoietic stem cells are derived from human umbilical cord blood cells.

In one embodiment, the stem and/or progenitor cells are purified to obtain CD34 ⁺ , CD38 ⁺ , CD90 ⁺ , CD45RA ⁺ , CD133 and/or CD49f ⁺ cells.

In another embodiment, the CD34+ cells are EPCR+ cells.

In a further embodiment, the NK cells are CD56 ⁺ or NKG2A ⁺ cells.

In another embodiment, the DC cells are CD11c ⁺ .

In one embodiment, the stem and/or progenitor cells are cultured with at least one cell expansion factor.

In another embodiment, the at least one cell expansion factor is interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), stem cell factor (SCF), interleukin-6 (IL-6), or a combination thereof.

In a further embodiment, the stem and/or progenitor cells are further cultured with an aryl hydrocarbon receptor (AHR) antagonist.

In one embodiment, the AHR antagonist is Stem Regenin1 (SR1) or CH223191.

In a preferred embodiment, the compound of formula I is

or a pharma- ceutically acceptable salt thereof.

In another embodiment, the compound of formula I is

It is the hydrobromide salt of

In a further embodiment, the compound of formula I is

or a pharma- ceutically acceptable salt thereof.

In another embodiment, the compound of formula I is

or a pharma- ceutically acceptable salt thereof.

In another embodiment, the stem and/or progenitor cells are expanded in a bioreactor.

In one embodiment, the patient is a human or an animal.

In another embodiment, the animal is a mouse.

In a further embodiment, a method of treating a viral infection in a patient or the use of a transplant of expanded stem and/or progenitor cells as described herein to treat a viral infection in a patient is also included.

In one embodiment, the viral infection is a CMV infection, an EBV infection, or an adenoviral cystitis.

In another embodiment, the DC cells and/or NK cells are expanded after 14 days, 21 days, 28 days, 56 days, 100 days, 6 months, 12 months, or 18 months in the patient's body.

In one embodiment, the inflammatory condition is controlled in the patient.

In one embodiment, the graft comprises a population of dendritic cells.

In another embodiment, the dendritic cell population is CD86 ⁺ CD34 ⁺ cells.

In a further embodiment, the graft comprises 40-50% dendritic cells.

In another embodiment, the graft comprises 1/3 of the immature dendritic cells.

In one embodiment, the graft comprises mast cells.

In another embodiment, the graft comprises approximately 10% mast cells.

In one preferred embodiment, the graft comprises FCER1 ⁺ CD34 ⁺ cells, CD34 ⁺ CD45RA ⁺ cells, CD34 ⁺ CD86 ⁺ cells, CD34 ⁺ CD45RA ^- cells and CD34 ^- cells.

Next, please refer to the attached drawing.

Figure 1 shows that UM171 promotes ex vivo expansion of undifferentiated dendritic cell precursors, in which cord blood-derived CD34+ cells were cultured in HSC expansion medium supplemented with vehicle (DMSO 0.1%), SR1 (500 nM) or UM171 (35 nM) for 7 and 14 days, and the frequency of dendritic cell precursors (iDCs: CD34+CD86+) was assessed by flow. FIG. 1 shows cell populations identified in cord blood samples expanded and cultured with DMSO, SR1 or UM171, demonstrating unique characteristics of the grafts. (A) Clinical trial design. (B) Patient cohort definition. (C) Cord blood availability using standard selection criteria for 70 kg patients. (D) Cord blood availability using criteria used for Cohort 2 patients. (E) Historical data at Hospital Maisonneuve-Rosemont showing relationship between CD34 cell dose (after thawing of CB units) and time to neutrophil engraftment. FIG. 1 shows measurements after NK cell engraftment in a UM171CB patient, with the arrow pointing to measurements approximately 2 months post-transplant. (A) Engraftment and chimerism: Time to engraftment of 100 neutrophils. (B) Engraftment and chimerism: Resolution of fever. (C) Engraftment and chimerism: Length of hospital stay for patients transplanted with UM171-expanded CB cells (blue) or unmanipulated CB cells (green). (D) Engraftment and chimerism: Chimerism by lineage at various time points (days) in patients transplanted with UM171-expanded CB. (E) Engraftment and chimerism: Comparison of CD4 counts at 3 and 12 months in patients transplanted with UM171-expanded CB vs. unmanipulated CB. (F) Engraftment and chimerism: IgG levels in recipients of UM171-expanded CB. Control CB: unmanipulated cord blood. FIG. 1 shows HLA matching of CB blood units in the UM171 patient cohort (left) and the contemporary CB cohort (right), showing the proportion of patients with 6-7/8 matches and 4-5/8 matches between patient/CB blood units. (A) Cumulative incidence of immunosuppressant discontinuation (B) Cumulative incidence of transplant-related mortality (TRM) (C) Kaplan-Meier estimates of overall survival (OS) in the UM171 expanded and unmanipulated CB cohorts.

The present disclosure provides a method for expanding natural killer cells, dendritic cells, or a combination thereof in vivo following transplantation of a cord blood graft expanded with UM171 or an analog thereof.

Therefore, there is provided a method of expanding dendritic (DC) cells and/or natural killer (NK) cells in vivo in a patient, comprising culturing with UM171 or an analog derived therefrom to generate a graft of expanded stem and progenitor cells prior to administration to the patient. The expansion or increase in dendritic (DC) and natural killer (NK) cell populations in the patient results in an increased immune response that reduces transplant-related mortality (TRM), severe graft-versus-host disease (GVHD), relapse, and/or severe viral infection.

Allogeneic hematopoietic stem cell (HSC) transplantation is the best available treatment to cure patients with blood cancers. Umbilical cord blood (CB) is the most attractive source of stem cells.

It is known that StemRegenin1 (SR1), a purine derivative that promotes ex vivo expansion of CD34 ⁺ cells, was able to expand HSCs but was unable to recapitulate long-term HSC expansion (Chen et al., 2012, Genes Dev., 26:2499-2511). A major concern with SR1 is its ability to support the growth of leukemic stem cells in vitro (Pabst et al., 2014, Nature Methods, 11:436-442).

Approximately 1% of CB cells express the CD34 surface antigen, which is composed of distinct subsets of stem and progenitor cells with different capacities to provide short-term or long-term hematopoiesis. These subsets differentially contribute to early and long-term recovery of mature blood cell production. Only long-term HSCs can provide lifelong hematopoietic reconstitution. In vitro expansion of CB-derived short-term HSCs (progenitor cells) dramatically shortens the time to neutrophil recovery after transplantation. However, most ex vivo cell expansion strategies described to date achieve this effect at the cost of losing long-term HSCs, thereby compromising sustained reconstitution and risking late graft failure. Thus, many of these strategies require a second CB transfusion to ensure long-term engraftment. In sharp contrast, as described and encompassed herein, the molecules used to amplify short-term repopulating cells derived from CBs can simultaneously expand, rather than deplete, long-term repopulating cells, paving the way for single-unit use of expanded CBs.

UM171, included herein (see U.S. Pat. No. 9,409,906, the contents of which are incorporated by reference), is a compound that has activity against undifferentiated cells that is rapidly reversible when the compound is removed from the culture system. UM171 does not independently induce cell proliferation in the absence of growth factors. UM171 does not promote mitosis but prevents cell differentiation. UM171 was tested in a fed-batch culture system. When mature cells are generated in CD34 ⁺ CB cultures, they release several negative cytokine regulators (Csaszar et al., 2012, Cell stem cell, 10:218-229). The fed-batch approach encompassed herein results in a reduction of endogenously produced negative regulatory factors. This system requires much less medium (culture system <1 liter vs. 10 liters in most studies) and better supports the maintenance of CB-derived HSCs. A 7-day culture is optimal to maximize cell quality. Importantly, fed-batch culture is a closed system that does not require cell manipulation, thus minimizing contamination risks and facilitating transition to cell product manufacturing.

As encompassed herein, the expansion of stem and/or progenitor cells can be carried out in a bioreactor, which can consist of any manufactured or engineered device or system that supports a biologically active environment, such as a culture of cells, for the expansion of said cells.

Therefore, after transplantation of the cord blood graft, the umbilical cord blood graft is treated with a compound having the general formula I defined herein:

or a salt or prodrug thereof,
each Y is independently selected from N and CH;
Z is -CN, -C(O)OR1, -C(O)N(R1)R3, -C(O)R1, or -heteroaryl (optionally substituted with one or more RA or R4 substituents), where (R1) and R3, when attached to a nitrogen atom, may be combined with the nitrogen atom to form a 3-7 membered ring which may contain one or more other heteroatoms selected from N, O and S, which ring is optionally substituted with one or more RA or R4;
W is -CN, -N(R1)R3, -C(O)OR1, -C(O)N(R1)R3, -NRC(O)R1, -NRC(O)OR1, -OC(O)N(R1)R3, -OC(O)R1, -C(O)R1, -NRC(O)N(R1)R3, -NR1S(O) ₂ R1, -benzyl (optionally substituted with 1, 2 or 3 RA or R1 substituents), -X-L-(X-L)n; -N(R1)R3, -X-L-(X-L)n-heteroaryl (optionally substituted with one or more RA or R4 substituents which are attached to either or both of the L and heteroaryl groups), -X-L-(X-L)n-heterocyclyl (optionally substituted with one or more RA or R4 substituents which are attached to either or both of the L and heterocyclyl groups), -X-L-(X-L)n-aryl (optionally substituted with one or more RA or R4 substituents), -X-L-(X-L) _n -NR1RA, or -(N(R1)-L) _n -N ⁺ R1R3R5R6- ^;
where n is an integer equal to either 0, 1, 2, 3, 4, or 5;
wherein when R1 and R3 are bonded to a nitrogen atom, R1 and R3 together with the nitrogen atom may form a 3- to 7-membered ring which may contain one or more other heteroatoms selected from N, O and S, and which ring is optionally substituted with one or more RA or R4;
Each X is independently selected from C, O, S, and NR1;
each L is independently -C1-6 alkylene, _-C2-6 alkenylene, _-C2-6 alkynylene, _-C3-7 cycloalkylene (optionally containing one or more other heteroatoms selected from N _, O and S), or _-C3-7 cycloalkenylene (optionally containing one or more other heteroatoms selected from N, O and S), wherein the alkylene, alkenylene, alkynylene, cycloalkylene and cycloalkenylene groups are each optionally and independently substituted with one or two R4 or RA substituents;
each R1 is independently -H, -C1-6 alkyl, _-C2-6 alkenyl, _-C2-6 alkynyl, _-C3-7 cycloalkyl, _-C3-7 cycloalkenyl, _-C1-5 perfluorinated group, -heterocyclyl, -aryl, -heteroaryl, or -benzyl, wherein the alkyl, alkenyl, alkynyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally and independently substituted with 1, ₂ , or 3 RA or Rd substituents;
R2 is -H, _{-Ci_6alkyl} (optionally substituted with one or more RA substituents), -C(O)R4, -L-heteroaryl (optionally substituted with one or more RA or R4 substituents), -L-heterocyclyl (optionally substituted with one or more RA or R4 substituents), or -L-aryl (optionally substituted with one or more RA or R4 substituents);
each R3 is independently -H, _{-C1-6 alkyl, -C2-6} alkenyl, _-C2-6 alkynyl _{, -C3-7} cycloalkyl, _-C3-7 cycloalkenyl, _-C1-5 perfluorinated group, -heterocyclyl, -aryl, -heteroaryl, or -benzyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally and independently substituted with 1, 2, or 3 RA or Rd substituents;
each R4 is independently -H, _{-C1-6 alkyl, -C2-6} alkenyl, _-C2-6 alkynyl _{, -C3-7} cycloalkyl, _-C3-7 cycloalkenyl, _-C1-5 perfluorinated group, -heterocyclyl, -aryl, -heteroaryl, or -benzyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally and independently substituted with 1, 2, or 3 RA or Rd substituents;
R5 is independently _-C1-6 alkyl, _{-C1-6 alkylene-C2-6} alkenyl (optionally containing one or more other heteroatoms selected from N, O and S), _-C1-6 alkylene- _C2-6 alkynyl (optionally containing one or more other heteroatoms selected from N, O and S), -L-aryl (optionally containing one or more RA or R4 substituents), -L-heteroaryl (optionally containing one or more RA or R4 substituents), _-C1-6 alkylene-C(O)O-, _-C1-6 alkylene-C(O)OR1, _-C1-6 alkylene-CN, _-C1-6 alkylene-C(O)NR1R3 (wherein R1 and R3 together with the nitrogen atom may form _a 3-7 membered ring which may contain one or more other heteroatoms selected from N, O and S), or -C1-6 alkylene-C(O)O-. _1-6 alkylene-OH;
R6 is halogen, —OC(O)CF ₃ , or —OC(O)R1;
Each RA is independently -halogen, -CF ₃ , -OR1, -L-OR1, -OCF ₃ , -SR1, -CN, -NO ₂ , -NR1R3, -L-NR1R1, -C(O)OR1, -S(O) ₂ R4, -C(O)N(R1)R3, -NRC(O)R1, -NRC(O)OR1, -OC(O)N(R1)R3, -OC(O)R1, -C(O)R4, -NHC(O)N(R1)R3, -NRC(O)N(R1)R3, or -N ₃
and
each Rd is independently -H, _-C1-6 alkyl, -C2-6 alkenyl _{, -C2-6} alkynyl, _-C3-7 cycloalkyl, _-C3-7 cycloalkenyl, _-C1-5 perfluorinated group, -benzyl, or -heterocyclyl;
The present invention encompasses methods of expanding natural killer cells, dendritic cells, or a combination thereof in vivo using the method.

In one embodiment, Z is —C(O)OR1, or -heteroaryl (optionally substituted with one or more RA or R1 substituents), R2 is H, —C _1-6 alkyl (optionally substituted with one or more RA substituents), or -L-aryl (optionally substituted with one or more RA or R4 substituents), and W is —N(R1)R3, where R1 is C _3-7 cycloalkyl substituted by RA and R3 is H.

In one embodiment, Z is -C(O)O-Ci- ₄ alkyl or 5 membered heteroaryl containing 2 to 4 heteroatoms (N or O); R2 is H, or -L-aryl (optionally substituted by halogen), OR1, Ci _-6 alkyl (optionally substituted by RA), -C(O)R4, -heterocyclyl, -C(O)OR4, or _C2-6 alkynyl; W is -N(R1)R3, where R1 is cyclohexyl substituted by RA and R3 is H.

In one embodiment, Z is COOMe, COOEt, tetrazole or oxadiazole.

In one embodiment, R2 is H, or -CH2-aryl (optionally substituted by halogen), OR1, _C1-6alkyl (optionally substituted by RA), -C(O)R4, -heterocyclyl, -C(O)OR4, or _C2-6alkynyl , where aryl is phenyl.

In one embodiment, R2 is H, --C _1-6 alkylene-heteroaryl, or --C _1-6 alkylene-aryl (optionally substituted with one or more RA or R4 substituents).

According to another embodiment, the compound is of formula I, IA or IIA, where Z is CO ₂ Me or 2-methyl-2H-tetrazol-5-yl;

R2 is benzyl or H,

W is NH-L-N(R1)R3, where L is _C2-4 alkylene or _C3-7 cycloalkylene and R1 and R3 are _C1-4 alkyl or H, or R1 and R3 together with the nitrogen atom to which they are attached form a 3-7 membered ring (which may contain one or more other heteroatoms selected from N, O and S), which ring is optionally substituted by one or more RA or R4.

According to another embodiment, the compound is a compound of formula I, where W is

It is.

The compounds of Formula I disclosed herein, including the representative compounds set forth below, including their preparation and characterization, are described in PCT Publication No. WO 2013/110198, the contents of which are incorporated by reference in their entirety, as well as in the Synthesis Methods section below.

As used herein, the term "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, C1-C6 in C1-C6 alkyl is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a straight-chain or branched saturated arrangement. Examples of C1-C6 alkyl as defined above include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, and hexyl.

As used herein, the term "cycloalkyl" is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms in the group. For example, C3-C7 in C3-C7 cycloalkyl is defined to include groups having 3, 4, 5, 6, or 7 carbons in a monocyclic saturated arrangement. Examples of C3-C7 cycloalkyl as defined above include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

As used herein, the term "alkenyl" is intended to mean an unsaturated straight or branched chain hydrocarbon group having the specified number of carbon atoms in the group, at least two of which are bonded together by a double bond, and having either E or Z regiochemistry and combinations thereof. For example, C2-C6 in C2-C6 alkenyl is defined to include groups having 2, 3, 4, 5, or 6 carbons in a straight or branched arrangement, and at least two of which are bonded together by a double bond. Examples of C2-C6 alkenyl include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, 1-butenyl, etc.

As used herein, the term "alkynyl" is intended to mean an unsaturated straight chain hydrocarbon group having the specified number of carbon atoms in the group, with at least two of the carbon atoms being joined together by a triple bond. For example, C2-C4 alkynyl is defined to include groups having 2, 3 or 4 carbon atoms in the chain, with at least two of the carbon atoms being joined together by a triple bond. Examples of such alkynyls include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

As used herein, the term "cycloalkenyl" is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms in the group. For example, C3-C7 in C3-C7 cycloalkenyl is defined to include groups having 3, 4, 5, 6, or 7 carbons in a monocyclic arrangement. Examples of C3-C7 cycloalkenyl as defined above include, but are not limited to, cyclopentenyl, cyclohexenyl, etc.

As used herein, the term "halo" or "halogen" means fluorine, chlorine, bromine or iodine.

As used herein, the term "haloalkyl" is intended to mean an alkyl as defined above, in which each hydrogen atom may be successively replaced by a halogen atom. Examples of haloalkyl include, but are not limited to, _CH2F , _CHF2 , and _CF3 .

As used herein, the term "aryl", alone or in combination with another group, means a carbocyclic aromatic monocyclic group containing six carbon atoms, which may be further fused to a second five- or six-membered carbocyclic group, which may be aromatic, saturated or unsaturated. Examples of aryl include, but are not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl, tetrahydronaphthyl, and the like. An aryl may be attached to another group at any suitable position on either the cycloalkyl ring or the aromatic ring.

As used herein, the term "heteroaryl" is intended to mean a monocyclic or bicyclic ring system of up to 10 atoms, in which at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Heteroaryls may be attached through a ring carbon atom or one of the heteroatoms. Examples of heteroaryls are thienyl, benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyrin, phenyl ... These include, but are not limited to, dinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl, isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl, thiazolo[4,5-b]-pyridine, tetrazolyl, oxadiazolyl, thiadiazolyl, thienyl, pyrimidoindolyl, pyridoindolyl, pyridopyrrolopyrimidinyl, pyrrolodipyridinyl, and fluorescein derivatives.

As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" is intended to mean a 3-, 4-, 5-, 6- or 7-membered non-aromatic ring system containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, piperidyl, 3,5-dimethylpiperidyl, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, tetrahydro-1H-thieno[3,4-d]imidazol-2(3H)-one, diazirinyl, and the like. Attachment to the ring may be at a nitrogen atom or a carbon atom of the ring, for example as described below.

As used herein, the term "optionally substituted with one or more substituents" or its equivalent "optionally substituted with at least one substituent" is intended to mean that the subsequently described circumstance event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. The definition is intended to mean from 0 to 5 substituents.

As used herein, the term "subject" or "patient" refers to humans and non-human mammals, such as primates, cats, dogs, pigs, cows, sheep, goats, horses, rabbits, rats, mice, etc.

If the substituent itself is not compatible with the synthetic methods described herein, the substituent can be protected by a suitable protecting group (PG) that is stable to the reaction conditions used in these methods. The protecting group can be removed at a suitable point in the reaction sequence of the method to obtain the desired intermediate or target compound. Suitable protecting groups and methods for protecting and deprotecting various substituents using such suitable protecting groups are well known to those skilled in the art. Examples can be found in T. Greene and P. Wuts, "Protecting Groups in Chemical Synthesis" (4th Edition), John Wiley & Sons, NY (2007), which is incorporated herein by reference in its entirety. Examples of protecting groups used throughout include, but are not limited to, Fmoc, Bn, Boc, CBz, and _COCF3 . In some cases, the substituent may be specifically selected to be reactive under the reaction conditions used in the methods described herein. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful in an intermediate compound in the methods described herein or is a desired substituent in a target compound.

As used herein, the term "pharmaceutically acceptable salts" refers to both acid addition salts and base addition salts.

As used herein, the term "pharmaceutical acceptable acid addition salt" is intended to mean an acid addition salt which retains the biological effectiveness and properties of the free base which are not biologically or otherwise harmful, such acid addition salts being formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

As used herein, the term "pharmacologically acceptable base addition salt" is intended to mean a base addition salt which retains the biological effectiveness and properties of the free acid, which are not biologically or otherwise undesirable. Such base addition salts are prepared by the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like.

The compounds encompassed herein, or pharma- ceutically acceptable salts thereof, may contain one or more asymmetric centers, chiral axes and chiral planes, and thus may give rise to enantiomers, diastereomers and other stereoisomers. They may be defined in terms of absolute stereochemistry, e.g., (R)- or (S)-, or, in the case of amino acids, (D)- or (L)-. The invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+)- and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques such as reverse-phase HPLC. Racemic mixtures may be prepared and then separated into the individual optical isomers, although these optical isomers may also be prepared by chiral syntheses. Enantiomers can be resolved by methods known to those skilled in the art, for example, by forming diastereomeric salts which can then be separated by crystallization, gas-liquid or liquid chromatography, or selective reaction of one enantiomer with an enantiomer-specific reagent. Those skilled in the art will appreciate that if the desired enantiomer is converted to another chemical entity by a separation technique, additional steps must then be performed to form the desired enantiomeric form. Alternatively, a specific enantiomer can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

Certain compounds encompassed herein may exist as a mixture of epimers, which means diastereoisomers that have the opposite configuration at only one of two or more stereocenters present in the respective compounds.

The compounds encompassed herein may exist in zwitterionic form, and the invention includes zwitterionic forms of these compounds and mixtures thereof.

Additionally, the compounds encompassed herein may exist in hydrated and anhydrous forms. Hydrates of the compounds of any of the formulas described herein are also included. In a further embodiment, the compounds according to any of the formulas described herein are monohydrates. In some embodiments, the compounds described herein contain about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less (by weight) of water. In other embodiments, the compounds described herein contain about 0.1% or more, about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, or about 6% or more (by weight) of water.

It may be convenient or desirable to prepare, purify, and/or handle the present compounds in the form of a prodrug. Thus, as used herein, the term "prodrug" pertains to a compound which, when metabolized (e.g., in vivo), yields the desired active compound. Typically, a prodrug is inactive or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic properties. Unless otherwise specified, a reference to a particular compound also includes its prodrug.

In one embodiment, compounds of formula I, more particularly UM171 or derivatives, can be used alone or in combination with AHR to differentiate monocyte-derived AML cell lines into immature and even mature functional DCs.

UM171 has the following structure:

Therefore, the following compounds are also included:

UM171 in a fed-batch system resulted in significant expansion of CD34 ⁺ CD45RA ⁻ cells, a key subpopulation encompassing all HSCs, including long-term repopulating cells (defined by their ability to reconstitute NSG mice 20-30 weeks after transplantation) and short-term repopulating cells such as CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte) precursors that determine time to neutrophil engraftment. CD34 ⁺ CD45RA ⁻ and phenotypes other than CD34 ⁺ are the best surrogate parameters for monitoring HSC expansion. Cells expanded in a fed-batch system with DMSO differentiate into mature cells with CD34 ⁺ expression dropping from 100% to less than 10% after 12 days.

As can be seen in Figure 1, when human CD34+ precursors were purified from cord blood samples and cultured with UM171 for 2 weeks, the compound promoted ex vivo enrichment of immature dendritic cell precursors, resulting in new grafts, as compared to SR1 for example (Figure 2). The cell populations expanded and generated upon exposure to other molecules (such as SR1) are different from those obtained with UM171. The dendritic cell population (CD86 ⁺ CD34 ⁺ ) constituted 40-50% of the graft in UM171 expanded cord blood, but less than 5% in SR1 expanded cord blood, and was undetectable in untreated cord blood. UM171 treatment dramatically altered the graft composition, resulting in a 500-fold increase in immature dendritic cells. Mast cells expressing FceR1A and c-Kit were also selectively expanded by UM171 exposure, occupying approximately 10% of the grafts (magnification >8000-fold).

The ability of UM171/fedbatch to expand short-term and long-term HSCs to unprecedented levels (expansion >1000-fold) results in rapid and sustained engraftment. Safety, feasibility, engraftment and immune reconstitution in patients are described here.

A cohort of 22 patients was tested for the potential of expanded CB units obtained with the UM171/fed-batch system to provide sustained engraftment with neutrophil recovery <21 days. Dose reduction was driven by a Wald test based on Cox modeling that takes into account the strong correlation between CD34 ⁺ infusion dose and neutrophil recovery within the clinically acceptable time point of 21 days. For safety reasons, a minimum of i) ^5x105 /kg live CD34 ⁺ cells and ii) ^1x106 /kg live CD3 ⁺ T cells must be infused. If these values could not be achieved or shipping criteria were not met, a second CB was infused (Figure 3A).

Cohort description and CB inclusion criteria before expansion are as follows:
Cohort 1: TNC ≥ 2.0 x ¹⁰⁷ /kg and CD34 ⁺ ≥ 1.0 x ¹⁰⁵ /kg;
Cohort 2: TNC > 1.5 x ¹⁰⁷ /kg and CD34 ⁺ > 0.5 x ¹⁰⁵ /kg, and Cohort 3: TNC > 1.25 x ¹⁰⁷ /kg and CD34 ⁺ > 0.25 x ¹⁰⁵ /kg.
To move from cohort 1 to cohort 2, a minimum of three patients had to achieve engraftment within 18 days of receiving a single unit of UM171-expanded CB with < ^2.0x105 cells/kg CD34+ at thaw. To move from cohort 2 to cohort 3, a minimum of three patients had to achieve engraftment within 18 days of receiving a single unit of UM171-expanded CB with < ^1.0x105 cells/kg CD34+ at thaw (Figure 3B). In cohort 2, 47% of banked CB units were available compared to only 5% using unexpanded CB (Figures 3C and 3D).

Based on the strong relationship between neutrophil engraftment and the dose of viable CD34 ⁺ cells infused, it is possible to estimate whether CD34 ⁺ cell dose reductions are safely achievable clinically in small cohorts of patients: for example, cell doses above 3× ¹⁰⁵ /kg of viable CD34 ⁺ cells predict very rapid engraftment, whereas levels below 0.5× ¹⁰⁵ /kg result in slower engraftment (Figure 3E).

Since then, 22 patients (numbered 1-22) have received a single unit of UM171 cord blood (N=22). Median follow-up was 18 months (range 1-28 months for patients 1-22). Results were compared to patients who received consecutive unmanipulated cord blood transplants at the same center (control CB). Transfusion of ^≥1x105 cells/kg of live CD34 ⁺ cells confirmed neutrophil engraftment within 21 days, and so this amount was transfused (Figure 3E). In addition, a CB-only control group was included that received the same conditioning regimen without ATG as the patients enrolled in this trial.

The CB selection process was standardized to avoid variability. The CBs selected for the study were compared with the CBs selected when patients were non-compliant with the protocol. This made it possible to demonstrate an improvement in HLA matching and a reduction in the need for double unit CBs.

CBs for expansion were purchased and shipped to manufacturing centers (FHCRC for the first 10 patients and CETC for the next 15 patients). CBs were thawed and subjected to selection of CD34 ⁺ cells. CD34 ⁺ cells were cultured in fed-batch with growth factors for 7 days, then washed, cryopreserved and shipped to the transplant center. CD34 ⁻ cells were cryopreserved and infused into patients at the time of transplant. All cell products were assessed for viability and phenotype at every step, and release criteria included sterility, mycoplasma, endotoxin, CD34 ⁺ expansion at least 10-fold, and viability >70%.

Patients received standard myeloablative conditioning (Barker et al., 2005, Blood, 105:1343-1347; Oran et al., 2001, Biology of blood and marrow transplantation: Journal of the American Society for Blood and Marrow Transplantation, 17:1327-1334). Expanded CB was infused on the day of transplantation, and CD4 products were infused the next day. GVHD prophylaxis consisted of mycophenolate mofetil and cyclosporine. Patients will be followed for a minimum of 3 years to ensure that no unexpected complications have occurred. Patients receive standardized supportive care.

As can be seen in Table 1 below, cell counts were measured at 14, 21, 28, 56, 100 days, 6 months, and 12 months after graft implantation. A panel of antibodies was selected to be measured to assess both myeloid and lymphoid lineage engraftment. Specifically:
- CD45 ⁺ CD3 ⁺ are lymphoid T cells. - CD56 ⁺ NKP46 ⁺ or NKG2A ⁺ are known NK cells.

Table 1 shows the number of cells measured per μl of blood, expressed as μl/blood ±5%.

As can be seen, a flare or expansion of NK cells was measured in vivo in patients between 21 days after transplantation and when numbers returned to their initial numbers at 12 months. As can be seen in Figure 4, the degree of NK cell expansion measured in patients at 2 months after engraftment (see arrows in Figure 4) is significantly higher than previously reported data (see Lucchini et al., 2015, Cytotherapy, 17:711-722).

A similar proliferation or expansion is observed when measuring the levels of two subsets of dendritic cells, CD11c ⁺ CD14 ⁺ cells and CD11c ⁺ CD14 ⁻ cells (Table 2).

The in vivo proliferation of natural killer and dendritic cells measured in patients transplanted with UM171-expanded cells has immunosuppressive activity, prevents GVH reactions, and allows graft tolerance. Immune cell recovery is faster with UM171-expanded cords, which is important for reducing post-hematopoietic cell transplant (post-HCT) infections and relapses. Thus, UM171 promotes the expansion of HSC and precursor cells by modulating pro- and anti-inflammatory responses and helping to control the inflammatory state.

The cumulative incidence of neutrophil recovery was 100%, with median times to 100 and 500 neutrophils of 9.5 (8-23) and 18 (10-30) days, respectively (Figure 5A). In control CB, the median time to 100 neutrophils was 11.5 days, whereas the time to 500 neutrophils was 19 days. Interestingly, achieving 100 neutrophils was independent of CD34 cell dose. There were no cases of late graft failure, and almost all patients achieved platelets ≥ 100, neutrophils ≥ 1500, and hemoglobin ≥ 100 at 6 and 12 months post-transplant. Rapid engraftment resulted in faster resolution of fever and shorter hospital stays after transplantation in UM171 patients (Figures 5B and 5C).

Chimerism analysis of various cell populations, particularly CD3, CD56, CD19, CD14 and CD33, revealed that the patient rapidly achieved 100% donor engraftment in all cell lines (Figure 5D).

After more than 10 months of follow-up, only two of 16 patients were reported to have grade III acute GVHD (both of which responded rapidly to steroids) and none to have moderate to severe chronic GVHD. Interestingly, viral infectious complications (e.g., CMV, EBV, adenovirus cystitis) were rare, indicating a robust immune response in these patients. Spontaneous resolution of EBV viremia was observed in two of three patients who were infected prior to transplantation (Table 3).

Thus, 11 patients had positive CMV serology and 6 developed viremia requiring treatment, but none developed CMV disease. Two cases of early-onset adenoviral cystitis occurring before D+25 required treatment with intravenous cidofovir. Three patients received rituximab for treatment of EBV viremia. No patients developed PTLD. Two cases of Pneumocystis pneumonia (PJP) occurred, the first patient was noncompliant due to prophylaxis for >2 months and the second patient was on atovaquone prophylaxis in a center where atovaquone-resistant PJP had occurred. There were two cases of dermatomal herpes zoster, both of which were off prophylaxis. No cases of invasive fungal infection, toxoplasmosis, or HHV6 infection were identified. Median CD4/μl at 3, 6, and 12 months were 196, 301, and 413, respectively (Figure 5E). Only one patient did not achieve a CD4 count of 50 by D+100. Median IgG at 3, 6, and 12 months were 5.9, 7.3, and 7.3 g/L, respectively (normal: 5.5-16.3) (Figure 5F).

Compared with the standard selection criteria for cord blood (TNC ≥ 2.0 x ¹⁰ /kg and CD34 ≥ 1.7 x ¹⁰ /kg), 12 of 22 patients had a better HLA match due to the lower minimum cell dose criteria. Thus, > 75% of patients received CB with HLA matching of 6-7/8 (5 5/8, 15 6/8, 2 7/8) (Figure 6). No patients were excluded from the trial because HLA-matched CB with sufficient cell dose could not be identified.

At 12 months after transplant, 85% of UM171CB patients had discontinued immunosuppressive therapy compared with 72% of control CB patients (Figure 7A). Overall, one patient died of diffuse alveolar hemorrhage, and the cumulative incidence of TRM at 1 year was 5% (95% CI: 1-31%) in the UM171CB cohort compared with 25% (95% CI: 12-54) in the control CB cohort (Figure 7B). OS at 12 months was 90% (95% CI: 67-98) compared with 75% (95% CI: 49-87) in the control CB (Figure 7C).

This study demonstrates that transplantation of a single-unit, smaller, more HLA-matched UM171-expanded CB into patients would reduce morbidity and mortality associated with CB transplantation. To this end, a Phase I-II clinical trial was conducted that demonstrated the primary endpoints of feasibility (expansion failure was observed in one cord, suspected to have an underlying anomaly), safety with a TRM of 5%, and rapid engraftment with the use of smaller, more HLA-matched cords.

Furthermore, rapid (<D+18), robust and sustained neutrophil engraftment was ensured without graft failure. Furthermore, the very early (D+10) recovery of 100 neutrophils resulted in rapid resolution of febrile neutropenia (D+7.5) and a shorter hospital stay (35 days for UM171CB vs. 47 days for CB controls), which was comparable to that seen with conventional allogeneic transplants (29.5 days for BM and 33 days for peripheral blood). The more rapid recovery of 500 neutrophils is expected to lead to earlier discharge of UM171 patients compared to BM-PB.

Cord blood has long been thought to be associated with poor and delayed T cell immune reconstitution, at least in part because the infused T cells are naive and because memory T cell deficiencies contribute to increased risk of viral infection. Despite the lack of memory T cell transfer in the expansion procedure described herein and a mean T cell loss rate of 33%, CD4 recovery is at least as rapid as unmanipulated CB transplants (Figure 4E). CD4 recovery has been shown to be important for preventing TRM and relapse as well as infectious complications. In pediatric CB transplantation, Admiraal et al. (2016, Blood, 128:2734-2741) reported that if CD4 counts of 50 were not achieved by D+100, the risk of relapse was 100% (vs. 24%) for AML, 31% (vs. 11%) for TRM, and 56% (vs. 73%) for OS. In the cohort of 22 patients described herein, only one patient did not achieve a CD4 count of 50 by D+100. Rapid immune recovery in this study led to no severe viral infections and no CMV disease or PTLD. Furthermore, two of three patients treated for EBV viremia had already improved EBV titers below the threshold before receiving rituximab.

The expected TRM in CB transplantation is higher than that in conventional transplantation because of slower neutrophil and immune reconstitution and higher risk of graft failure. High frequency of TRM and longer hospital stay are the main reasons for the decline in interest in CB and the increasing use of haploidentical transplantation. HCT-CI is the best validated index to predict TRM in transplantation. TRM with CB has been reported to be 26% and 50% for HCTCI <3 and ≥3, respectively. The median HCT-CI in this study was 2, with 8 patients (38%) with a score ≥3. Thus, a TRM of 5% is considered to be quite low. There are many reasons that explain and support the low TRM: i) rapid neutrophil recovery at D+18, reaching 100 neutrophils very quickly, ii) better HLA matching, with >75% of patients receiving CBs with ≥6/8 alleles, iii) very low risk of graft failure, iv) no use of ATG, v) low risk of severe viral infections due to rapid immune reconstitution, and vi) no steroid-refractory GVHD. TRM with CB transplantation beyond 12 months is rare due to the low incidence of chronic GVHD, and therefore the TRM described herein is unlikely to increase dramatically with longer follow-up.

In summary, single-unit UM171-expanded cord blood transplants have shown excellent results in patients with high-risk hematologic malignancies and multiple comorbidities. The findings provided herein confirm that single-unit UM171-expanded CB is an acceptable graft when an HLA-matched donor is unavailable.

While the present disclosure has been described in connection with certain embodiments thereof, it will be understood that the disclosure is capable of further modifications, and that this application is intended to cover all such variations, uses, or applications, including departures from the present disclosure, e.g., as come within known or customary practice in the art and as may fall within the essential characteristics hereinbefore and as set forth in the appended claims.

Claims (29)

1. A graft of expanded stem and/or progenitor cells cultured with at least one compound for use in a method for expanding dendritic (DC) cells in vivo in a patient, wherein the stem and/or progenitor cells are human hematopoietic stem cells (HSC), and the at least one compound is a compound represented by formula I:
or a salt thereof,
each Y is independently selected from N and CH;
Z is,
- C.N.,
-C(O)OR1,
-C(O)N(R1)R3,
-C(O)R1, or -heteroaryl (optionally substituted with one or more RA or R4 substituents).
and
wherein when (R1) and R3 are bonded to a nitrogen atom, (R1) and R3 together with said nitrogen atom may form a 3- to 7-membered ring which may contain one or more other heteroatoms selected from N, O and S, and said ring may be substituted with one or more RA or R4;
W is
- C.N.,
-N(R1)R3,
-C(O)OR1,
-C(O)N(R1)R3,
-NR1C(O)R1,
-NR1C(O)OR1,
-OC(O)N(R1)R3,
-OC(O)R1,
-C(O)R1,
-NR1C(O)N(R1)R3,
-NR1S(O) _2R1 ,
-benzyl (optionally substituted by 1, 2 or 3 R or R substituents);
-X-L-(X-L)n-N(R1)R3,
-XL-(XL)n-heteroaryl, optionally substituted with one or more RA or R4 substituents attached to either or both of the L and heteroaryl groups;
-XL-(XL)n-heterocyclyl (optionally substituted with one or more RA or R4 substituents attached to either or both of the L and heterocyclyl groups);
-XL-(XL)n-aryl (optionally substituted by one or more R or R substituents);
-X-L-(X-L) _n -NR1RA, or -(N(R1)-L) _n -N ⁺ R1R3R5R6 ^-
and
where n is an integer equal to either 0, 1, 2, 3, 4, or 5;
wherein when R1 and R3 are bonded to a nitrogen atom, R1 and R3 together with said nitrogen atom may form a 3- to 7-membered ring which may contain one or more other heteroatoms selected from N, O and S, said ring being optionally substituted with one or more RA or R4;
Each X is independently selected from O, S, and NR1;
Each L is independently
_-C1-6 alkylene,
_-C2-6 alkenylene,
_-C alkynylene,
-C _3-7 cycloalkylene (which may contain one or more other heteroatoms selected from N, O and S), or -C _3-7 cycloalkenylene (which may contain one or more other heteroatoms selected from N, O and S).
and
wherein the alkylene, alkenylene, alkynylene, cycloalkylene, and cycloalkenylene groups are each optionally independently substituted with one or two R or RA substituents;
Each R1 is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-heterocyclyl,
-aryl,
-heteroaryl, or -benzyl,
wherein the alkyl, alkenyl, alkynyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally and independently substituted with 1, 2 or 3 Rd substituents;
R2 is,
-H,
-C _1-6 alkyl (optionally substituted by one or more RA substituents),
-C(O)R4,
-L-heteroaryl (optionally substituted by one or more R or R substituents);
-L-heterocyclyl (optionally substituted with one or more RA or R4 substituents), or -L-aryl (optionally substituted with one or more RA or R4 substituents).
and
Each R3 is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-heterocyclyl,
-aryl,
-heteroaryl, or -benzyl,
wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally substituted with 1, 2 or 3 R or R substituents;
Each R4 is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-heterocyclyl,
-aryl,
-heteroaryl, or -benzyl,
wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, perfluorinated alkyl, heterocyclyl, aryl, heteroaryl and benzyl groups are each optionally substituted with 1, 2 or 3 R or R substituents;
Each R5 is independently
-C _1-6 alkyl,
_{-Ci_6} alkylene _{-C2_6} alkenyl (which may contain one or more other heteroatoms selected from N, O and S);
_{-Ci_6} alkylene _{-C2_6} alkynyl (which may contain one or more other heteroatoms selected from N, O and S);
-L-aryl (optionally containing one or more R or R substituents);
-L-heteroaryl (optionally containing one or more R or R substituents);
—C _1-6 alkylene-C(O)O—,
_-C1-6 alkylene-C(O)OR1,
—C _1-6 alkylene-CN,
_-C1-6 alkylene-C(O)NR1R3, where R1 and R3 together with the nitrogen atom may form a 3-7 membered ring which may contain one or more other heteroatoms selected from N, O and S, or _-C1-6 alkylene-OH
and
R6 is,
-halogen,
-OC(O) _CF3 , or -OC(O)R1
and
Each RA is independently
-halogen,
_-CF3 ,
-OR1,
-L-OR1,
_-OCF3 ,
-SR1,
- C.N.,
_-NO2 ,
-NR1R3,
-L-NR1R1,
-C(O)OR1,
-S(O) _2R4 ,
-C(O)N(R1)R3,
-NR1C(O)R1,
-NR1C(O)OR1,
-OC(O)N(R1)R3,
-OC(O)R1,
-C(O)R4,
-NHC(O)N(R1)R3,
-NR1C(O)N(R1)R3, or _-N3
and
Each Rd is independently
-H,
-C _1-6 alkyl,
_-C2-6 alkenyl,
_-C alkynyl,
-C _3-7 cycloalkyl,
_-C cycloalkenyl,
_-C1-5 perfluorinated group,
-benzyl, or -heterocyclyl.
That is, a graft. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use as described in claim 1, wherein said expansion of DC cells stimulates an immune response in said patient. A transplant of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use as claimed in claim 1 or 2 to reduce severe graft-versus-host disease (GVHD), relapse, and/or severe viral infection. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use as claimed in claim 1, wherein said hematopoietic stem cells are derived from umbilical cord blood cells, mobilized peripheral blood cells or bone marrow cells. A transplant of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use as claimed in claim 4, wherein said hematopoietic stem cells are derived from human umbilical cord blood cells. A graft of expanded stem and/or progenitor cells cultured ^with at ^least one compound, said at least one compound having formula I, for use according ^to any one of claims 1 to 5, wherein the stem and progenitor cells are purified to obtain CD34 ⁺ , CD38+, CD90+, CD45RA+, CD133 and/or CD49f ⁺ cells. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use as claimed in claim 6, wherein said CD34+ cells are EPCR+ cells. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 7 , wherein said DC cells are CD11c ⁺ cells. 9. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 8 , wherein said stem and/or progenitor cells are cultured with at least one cell expansion factor. 10. The transplant of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to claim 9 , wherein said at least one cell expansion factor is interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), stem cell factor (SCF), interleukin-6 (IL-6), or a combination thereof. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 10 , wherein said stem and/or progenitor cells are further cultured with an aryl hydrocarbon receptor (AHR) antagonist. The use of claim 11 , wherein the AHR antagonist is Stem Regenin1 (SR1) or CH223191, the transplant of expanded stem and/or progenitor cells cultured with at least one compound, the at least one compound having formula I. The compound of formula I is UM171
13. The method of claim 1 , wherein the at least one compound has formula I, or a pharma- ceutical acceptable salt thereof, is administered to a subject in need of treatment with an inflammatory cytokine such as erythrocytes or cerebrospinal fluid (ICF) or a pharma- ceutical acceptable salt thereof. The compound of formula I is
14. The use of any one of claims 1 to 13 , wherein the at least one compound has formula I, is the hydrobromide salt of the compound. The compound of formula I is
15. The method of claim 1 , wherein the at least one compound has formula I, or a pharma- ceutical acceptable salt thereof. The compound of formula I is
16. The method of claim 1 , wherein the at least one compound has formula I, or a pharmaceutically acceptable salt thereof. 17. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 16 , wherein said stem and progenitor cells are expanded in a bioreactor. A transplant of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 17 , wherein the patient is a human or an animal. 20. The method of claim 18 , wherein the animal is a mouse, the explant of expanded stem and/or progenitor cells cultured with at least one compound, the at least one compound having formula I. 20. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 19 for treating a viral infection in said patient. 21. The use of claim 20 , wherein the viral infection is a CMV infection, an EBV infection or an adenoviral cystitis, the transplant of expanded stem and/or progenitor cells cultured with at least one compound, the at least one compound having formula I. 22. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 21 , wherein an inflammatory condition is controlled in said patient. 23. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 22 , wherein said graft comprises a population of dendritic cells and mast cells. A transplant of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use as described in claim 23 , wherein the dendritic cell population is CD86 ⁺ CD34 ⁺ cells. 25. The use of claim 23 or 24 , wherein the graft comprises 40-50% dendritic cells in an amount of 40-50%. 26. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 25 , wherein said graft comprises one-third of immature dendritic cells. 27. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use according to any one of claims 1 to 26 , wherein the graft comprises mast cells. 28. A graft of expanded stem and/or progenitor cells cultured with at least one compound, said at least one compound having formula I, for use as described in claim 27 , wherein said graft comprises mast cells amounting to about 10%. 29. The graft ^of expanded stem and/or ^{progenitor cells} cultured with ^{at least} one compound, said at least one compound having formula I, for use according to any one of claims 1 to 28 , wherein said graft comprises FCER1 ⁺ CD34 ⁺ cells, CD34+CD45RA ⁺ cells, CD34+CD86+ cells, CD34 ⁺ CD45RA- cells and CD34- cells.